Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism

Summary Placental hormones orchestrate maternal metabolic adaptations to support pregnancy. We hypothesized that placental ER stress, which characterizes early-onset pre-eclampsia (ePE), compromises glycosylation, reducing hormone bioactivity and these maladaptations predispose the mother to metabolic disease in later life. We demonstrate ER stress reduces the complexity and sialylation of trophoblast protein N-glycosylation, while aberrant glycosylation of vascular endothelial growth factor reduced its bioactivity. ER stress alters the expression of 66 of the 146 genes annotated with “protein glycosylation” and reduces the expression of sialyltransferases. Using mouse placental explants, we show ER stress promotes the secretion of mis-glycosylated glycoproteins. Pregnant mice carrying placentas with junctional zone-specific ER stress have reduced blood glucose, anomalous hepatic glucose metabolism, increased cellular stress and elevated DNA methyltransferase 3A. Using pregnancy-specific glycoproteins as a readout, we also demonstrate aberrant glycosylation of placental proteins in women with ePE, thus providing a mechanistic link between ePE and subsequent maternal metabolic disorders.


INTRODUCTION
Successful pregnancy requires fine-tuned sharing of resources between mother and fetus to support proper growth of the fetal-placental unit without compromising maternal health and survival. 1,2 Extensive maternal adaptations ensure an adequate supply of nutrients and oxygen to the growing fetus. There are also fetal adaptations and all these adaptations are sometimes described as maternal-fetal conflict. They include increased peripheral insulin resistance to mobilize glucose, amino acids, and lipids for transfer, and cardiovascular changes. 3,4 These adaptations are largely orchestrated by placental hormones. Increasing evidence indicates that pregnancies complicated by placental dysfunction, including fetal growth restriction (FGR) and/or pre-eclampsia (PE), adversely influence the woman's long-term health, increasing the risk of developing cardiovascular disease, type II diabetes, and obesity in later life 3-to 10-fold. [5][6][7][8] The placenta plays a central role in maternal-fetal resource allocation through monitoring of maternal nutrient availability. 2 Maternal energy reserves are laid down during early to mid-gestation and mobilised to support rapid fetal growth during the second half. 2 Increased maternal appetite and leptin resistance initially promote lipid accumulation, 2 while developing maternal peripheral insulin resistance later increases hepatic gluconeogenesis, reduces glucose uptake in maternal skeletal muscle and adipose tissue, and increases lipolysis, releasing glucose and lipids for transfer to the fetus. 9,10 The liver plays an essential role in glucose homeostasis by regulating various glucose metabolic pathways. 11 Maternal hepatic physiology and function adjust accordingly as pregnancy progress, including increased cell proliferation and mass, 12 and elevated basal endogenous glucose production by 30%. 13 However, in pre-eclamptic pregnancies, these changes are either impaired or exacerbated. 14,15 A number of placentally derived factors (including placental growth hormone variant, placental lactogen, adiponectin, and insulin-like growth factor-binding proteins) contribute to the maternal metabolic changes and concentrations of these factors increase throughout pregnancy. 2,16 The majority are synthesized in the rough ER where folding and glycosylation of nascent polypeptides are also affected. Protein glycosylation links oligosaccharides side chains or glycans to the nitrogen (N-glycans) and oxygen (O-glycans) of asparagine and serine/threonine respectively. N-linked glycosylation is initiated in the ER and completed in the Golgi complex. A family of glycosyltransferases and glycosidases regulates the formation of glycans in a stepwise fashion, influenced by substrate availability, gene transcription, enzyme activity, and enzyme location within the organelles. 17 The process is completed by sialic acid end-capping, protecting glycoproteins from systemic clearance. 18 Therefore, correct glycosylation is essential for circulating protein half-life, receptor affinity and antigenicity. [18][19][20] Cells and organs suffering from ER stress secrete aberrantly glycosylated proteins 21,22 which been closely linked to various diseases. 17 We have previously shown the presence of ER stress in placentas from pregnancies complicated by earlyonset pre-eclampsia (ePE), FGR, and gestational diabetes. 23,24 Consequently, we hypothesize that misglycosylation may compromise the activity and bioavailability of placental factors, thereby adversely affecting maternal physiological adaptations, placing stress on organ systems and potentially increasing susceptibility to the future development of metabolic diseases. To address this hypothesis, we investigated whether placental ER stress altered the function of placentally derived factors and perturbed maternal physiological adaptation. We first used an in vitro cell model and a novel ex vivo junctional zone (Jz) placental explant model to determine whether ER stress can indeed alter the glycosylation of placentally derived glycoproteins. We used transcriptome analysis and a functional bioassay to investigate the molecular mechanisms and consequences of mis-glycosylation. We subsequently generated a transgenic mouse model with placental junctional zone-specific deletion of Perk (Jz-Perk À/À ). We examined whether chronic ER stress generated in the junctional zone (the region of the placenta responsible for endocrine activity) perturbs maternal physiology, focusing on maternal hepatic glucose metabolism. Finally, we sought evidence for aberrantly glycosylated proteins in samples from human pregnancies complicated by early-onset pre-eclampsia, focusing on pregnancy-specific glycoproteins (PSGs) that are secreted largely by the placenta, although low levels of the PSGs are also expressed in the gastrointestinal tract but at levels several orders of magnitude lower. 25

Endoplasmic reticulum stress alters glycosylation pattern of secretory proteins
To investigate whether ER stress underpins the secretion of mis-glycosylated placental glycoproteins, we treated trophoblast-like BeWo cells in the serum-free medium with thapsigargin (Tg), an ER stress inducer and sarco/ER Ca 2+ -ATPase inhibitor, following by glycomic analysis of conditioned media. Primary trophoblasts were not used because their isolation and culture induce substantial ER stress that would likely confound the data. 26 The N-glycans in the conditioned medium were released from the glycoproteins and analyzed by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry. N-linked glycan structures can be classified into three major subtypes based on their degree of processing: oligomannose, hybrid, and complex. As shown in Figure S1, both control and Tg-treated samples contained a diverse mixture of oligomannose, hybrid, and complex N-glycans. In untreated conditioned medium, the proportions of glycans with oligomannose, hybrid, and complex structures were 26%, 5%, and 69% respectively. In comparison, under ER stress conditions, these ratios shifted to 51%, 19%, and 30% respectively ( Figure 1A). In addition, ER stress reduced the sialylation of complex N-glycans indicated by a decrease in the relative abundance of fully sialylated species (m/z 3776) in comparison to partially and un-sialylated species (m/z 2693, 3054, 3415) (blue peaks inside the blue shaded area in Figure S1). These results are consistent with the study by Wong et al. 22 We did not detect any glycan structures containing N-Glycolylneuraminic acid (NeuGc), which is only found in non-human mammals. As humans are unable to synthesize this (due to a mutation in CMP-N-acetylneuraminic acid hydroxylase) this suggests that possible contamination with FBS is low.
While mass spectrometry gives an overview of a shift in the overall composition of the glycome within a sample, it does not allow the direct comparison of the absolute abundance of any specific glycan between two samples. We, therefore, sought to use a placental-specific glycoprotein as an example to investigate the overall change of degree of glycosylation in the protein under ER stress. Human chorionic gonadotrophin (hCG), one of the principal secretions of trophoblast, contains two heavily N-glycosylated subunits iScience Article a and b. 27 We used Western blotting analysis because the degree of glycosylation and/or the complexity of glycan structures can be detected by the pattern, intensity and molecular weights of hCG band(s). Following treatment with Tg or another ER stress inducer, tunicamycin (Tm) (which inhibits N-glycoslyation and therefore can be used as a positive control) glycosylation of the secreted hCGb chain was markedly decreased ( Figure 1B). The effect was especially prominent following Tg treatment when little glycosylated hCG was observed. This suggests that ER stress not only impairs the complexity of glycans being synthesized but also greatly reduces the overall level of protein glycosylation.
A D E C B Figure 1. ER stress alters protein glycosylation and function BeWo cells, grown in serum-free medium were treated with ER stress inducers, thapsigargin (Tg) (500 nM) or tunicamycin (Tm) (1 mg/mL) for 24 h. Conditioned media were harvested and cell lysates prepared for glycomic or Western blotting analysis. (A) Glycomic analysis of the trophoblastic BeWo cell secretome. N-glyans were released from the secreted protein and analyzed by MALDI-TOF MS. The pie charts summarize the distribution of three glycan subtypes: oligomannose, hybrid, and complex structure glycans in secreted glycoproteins from untreated control and Tg-treated BeWo cells respectively. The pie charts were plotted from the average value of 2 independent experiments. (B) ER stress reduces the overall glycosylation of secreted hCG. Western blotting for hCG b chain in conditioned media and cell lysates prepared from BeWo cells treated with Tm or Tg. (C-E) ER stress-induced aberrant glycosylation of VEGFA reduces its bioactivity. (C) Cell lysates and conditioned media were collected from BeWo cells after Tg or Tm treatment for Western blot analysis of level of cellular and secreted VEGFA. (D-E) Alteration of glycosylation and loss of bioactivity in secreted VEGFA by Eif2s1 tm1RjK mutant MEFs. (D) Eif2s1 tm1Rjk homozygous mutant MEFs showed no change in Vegfa transcripts (detected by RT-PCR) and cellular protein but an altered band pattern of secreted VEGFA (red arrow). Western blot was used to visualize cellular VEGFA and secreted VEGFA isoforms in cell lysates and conditioned media. (E) VEGFA secreted by Eif2s1 tm1Rjk homozygous mutant MEFs fails to activate KDR/VEGFR2 in endothelial cells. Human umbilical vascular endothelial cells (HUVECs) were pre-treated with serum-free medium for 6 h before challenge with conditioned media, 25 ng/mL recombinant Vega, or control medium for 10 min. Anti-P-VEGFR2 antibody was used to detect the activation of VEGFR2. Ponceau S staining showed equal protein loading among samples. wt, wild type fibroblasts; Mut, Eif2s1 tm1RjK mutant fibroblasts; P-VEGFR2, phosphorylated VEGFR2; PM, protein marker. The changes in complexity and amount of glycosylation may perturb the function and activity of secreted glycoproteins. To test this, we selected vascular endothelial growth factor A (VEGFA) as an exemplar in preference to hCG to assess the effects of ER stress on protein activity. Bioassays for hCG typically use fresh testicular cells collected from mice or rats. The complexity and the use of live animals make this bioassay unattractive. In contrast, VEGFA rapidly induces the phosphorylation of VEGF receptor 2 (VEGFR2) and thus the activity of VEGFA can be evaluated directly using readily available human umbilical vein endothelial cells (HUVECs). This cell-based bioassay is rapid and highly specific for VEGFA. The treatment of BeWo cells with Tg and Tm altered the glycosylation profiles of different isoforms of VEGFA in the conditioned medium (CM) ( Figure 1C, black arrows) confirming that ER stress alters the pattern and amount of glycosylation of secreted glycoproteins. However, the conditioned medium could not be used directly in an assay as residual Tg and Tm in the conditioned medium confound this experiment. Since VEGFA also expressed by human trophoblast 28 and by mouse embryonic fibroblasts (MEFs) a genetic model of ER stress can be used which avoids interference from any residual ER stress inducer present. Human VEGFA and mouse VEGFA are highly homologous (83.4% protein sequence identity) and both contain a single glycosylation site (UniportKB-Q00731). We, therefore, used a genetic model of ER stress, Eif2s1 tm1RjK in which serine 51 is replaced by alanine in eIF2a resulting in continuous ER stress in homozygous mutant mouse embryonic fibroblasts (MEFs). 29, 30 It is worth noting that Tg leads protein misfolding through the disruption of ER Ca 2+ homeostasis while in the Eif2s1 tm1RjK mutant cells, the high protein translation rate overwhelms the protein folding machinery resulting in protein misfolding. Although their precise effects on the protein glycosylation profile are likely to be different, the aberrant glycosylation in both conditions is likely to have a similar consequence on the protein function/activity. We first determined levels of Vegfa transcripts and cellular protein by RT-PCR and Western blotting. There was no difference between wild-type (wt) and Eif2s1 tm1RjK mutant (Mut) MEFs ( Figure 1D). Despite no change in cellular VEGFA protein level, we observed an increase in the highest molecular weight VEGFA and an extra band of VEGFA in CM from the mutant MEFs ( Figure 1D, red arrow). To evaluate the biological activity of the VEGFA in the CM we briefly exposed human umbilical vascular endothelial cells (HUVECs) to CM harvested from either wt or Mut MEFs. As expected, both wt CM and recombinant VEGFA (rVEGFA) induced rapid phosphorylation of VEGFR2. However, CM from the Mut MEFs failed to do so, indicating the loss of VEGFA bioactivity (Figure 1E). We have previously reported that conditioned medium from Eif2s1 tm1RjK mutant MEFs failed to maintain trophoblast stem cells in a pluripotent state, 30 consistent with loss of functional activity of other secreted growth factors.

Endoplasmic reticulum stress modulates the expression of genes involved in glycan biosynthesis
We next investigated the mechanisms by which ER stress influences protein glycosylation using RNA-Seq. We generated RNA-Seq datasets from BeWo cells treated with and without Tg (5 independent replicate pairs). Read numbers and alignment rates are given in Table S1. Principal Component Analysis (PCA) showed two distinct groups of samples with separation dependent on Tg treatment ( Figure S2A). Differential expression analysis using DESeq2 showed there were 1712 and 1482 transcripts increased and decreased respectively following Tg treatment (P adj < 0.05 and absolute fold-change R2, Table S2; differentially expressed genes, DEGs).
To investigate how Tg affects biological processes in an unbiased manner we used gprofiler2 to identify over-represented GO terms related to up-and down-regulated genes. Terms associated with the cell cycle and DNA replication were over-represented in the down-regulated genes. For example, ''cell cycle DNA replication,'' (GO:0044786, P adj = 8.4 3 10 À11 ). Among the up-regulated genes, many terms related to ER stress were enriched, for example ''response to ER stress'' GO:0034976, P adj = 5.2 3 10 À20 (Figure 2A). Many of the most differentially regulated genes are annotated with these or related terms as highlighted in the heatmap of the top 100 DEGs ordered by P adj (from smallest to largest) ( Figure S2B). Furthermore,  Figure S3A; Tables S3 and S4).
Having observed changes in the glycan profile of the conditioned media ( Figures 1A and 1B; S1), we noted Tg treatment also changed the transcript levels of genes involved in protein glycosylation. In the GSE analysis GOBP_ER_Mannose_Trimming (NES = À2.05, p = 0.000 and FDR q = 0.002) and GOBP_Protein_Deglycosylation (NES = À1.97, p = 0.000 and FDR q = 0.008) were ranked 17 th and 21 st respectively indicating the alteration of protein glycosylation upon ER stress ( Figure 2B). These gene sets are listed as heatmaps and tables ( Figure S3B; Tables S5 and S6).
146 genes associated with the GO:BP term ''Protein glycosylation,'' GO:0006486, are present in the dataset and 66 were identified as differentially regulated by Tg treatment (P adj < 0.05 and absolute fold-change R1.5, Table S7). This is very significant over-representation (Fisher exact test, two-sided, p = 2.2 3 10 À16 and the genes are shown in a heatmap ( Figure 2C). Sialyltransferases involved in N-linked glycosylation include ST3GAL4 & ST3GAL6 for a2,3 linkages; ST6GAL1 and ST6GAL2 for a2,6 linkages and ST8SIA2 and ST8SIA4 for a2,8 linkages. 31 The mRNAs encoding ST3GAL4, ST3GAL6, ST6GAL1, and ST8SIA4 were also reduced in Tg-treated BeWo cells ( Figure 2D) while ST6GAL2 and ST8SIA2 mRNAs had very low (<4) and no counts respectively. This would explain the loss of sialylation observed in the glycomic analysis above ( Figure S1). Changes in genes involved in N-glycosylation are illustrated in the KEGG pathway for N-glycan biosynthesis (KEGG: 00510, Figure S4).

Endoplasmic reticulum stress induces placental tissues to release aberrantly glycosylated proteins
To determine whether these cell culture-based findings occur in the placenta in vivo, we established a novel mouse placental junctional zone explant culture ex vivo model. The mouse placenta has two distinctive regions, the junctional zone (Jz) for endocrine activity and labyrinthine zone (Lz) for nutrient and gaseous exchange. The Jz is an active secretory tissue, confirmed by immunochemical staining of the ER chaperone GRP78 which is almost exclusively localized to the Jz ( Figure S5A). We dissected the placenta into decidua, Jz and Lz ( Figure S5B) and estimated the purity by RT-qPCR using Jz and Lz marker genes (Tpbpa and Gcm1 respectively). Dissected Jz was contaminated with Lz (11.9% G 4.4%, mean G SD) and dissected Lz contaminated with Jz (1.2% G 0.4%, mean G SD, n = 4, Figure S5C). We assessed the viability of the explants after 48 h in culture using the MTT assay which confirmed the presence of functional mitochondria ( Figure S5D). Furthermore, phosphorylated AKT (a cell survival and proliferation kinase) was increased whereas levels of ER stress markers (P-EIF2a and ATF4), and phosphorylation of stress kinases JNKs and energy sensing kinase AMPKa were lower than observed at t = 0 ( Figure S5E).
Treatment with Tg (100 nM) induced mild ER stress in Jz explants, causing a 2.6-fold (p = 0.005) increase in ATF4, while GRP78 and XBP1 remained unchanged after 48 h, indicating the activation of low-grade ER stress ( Figure 3A). Both the study from Wong et al. 22 and our study (Figures 1A and S1) have shown that Tg treatment alters the glycosylation pattern of secretory proteins -increasing glycans with oligomannose while decreasing glycans with sialyation on specific N-glycan epitopes. This would lead to a change of molecular weight and isoelectric focusing point (pI) of the mis-glycosylated glycoprotein. Therefore, we analyzed proteins in conditioned media from both untreated and Tg-treated Jz explants using two-dimension fluorescence Difference Gel Electrophoresis (DIGE). Nineteen of the differentially expressed spots (p < 0.05, Figure S5F) were selected, excised, digested with trypsin, and identified by LC-MS/MS proteomic analysis. Eighteen of the spots were clustered closely together, indicating likely changes in glycan structure. The identities of these spots are listed in Figure S5G. Indeed, among these were two clusters containing 8 and 3 spots which we identified as two members of the Carcinoembryonic Antigen-Related Cell Adhesion Molecules (CEACAM) family, (CEACAM11 and CEACAM12). The murine CEACAM family is closely related to the human pregnancy-specific glycoprotein (PSG) family. 32 Both CEACAM11 and CEACAM12 contain 4 glycosylation sites and are synthesized and secreted by the mouse placenta. 32 We detected Tg-induced changes in molecular weight and pl ( Figure 3B, red and blue circles) with changes in the spot intensity ratios. For example, for CEACAM11, spots 1503 and 1553 increased by 13% (p = 0.06) and 11.8% (p = 0.02) while spots 1549 and 1546 decreased by 16.5% (p = 0.046) and 20.4% (p = 0.027) respectively ( Figure 3C). Spots with reduced molecular weight reflect a reduction in glycan complexity while spots with increased pI suggested potential loss of sialic acid endcap(s). For instance, the Tg-mediated decrease in CEACAM11 spots 1549 and 1546 was associated with ll OPEN ACCESS 6 iScience 26, 105911, January 20, 2023 iScience Article increased protein level of spot 1553, which has a slightly reduced molecular weight and a higher pI, suggesting potential loss of sialic acid(s). These examples further support the hypothesis that ER stress in placental tissue can cause the secretion of aberrantly glycosylated proteins. Therefore, we next investigated whether ER stress-mediated perturbation of placental endocrine function affects maternal physiological adaptations to pregnancy.

Jz-specific endoplasmic reticulum stress reduces placental efficiency and increases embryonic lethality
We generated a placental-specific ER-stress mouse model in which Perk (PRKR-like ER kinase) that encodes one of the three sensors in the ER unfolded protein response (UPR) signaling pathways is selectively ablated in the junctional zone (Jz-Perk À/À ) using Cre recombinase driven by the Tpbpa promotor. This is active in ectoplacental cone (EPC) cells starting between embryonic days E7.5 and 8.5, and later in the spongiotrophoblast layer of the mature placenta. EPC-derived cells include spiral artery trophoblast giant cells (TGCs), canal TGCs, and trophoblast glycogen cells as well as the more numerous spongiotrophoblast cells. 33 However, it is the spongiotrophoblast cells that have high secretory activity as demonstrated by strong staining for the ER chaperone GRP78. The trophoblast glycogen cells in the Jz do not stain for this marker ( Figure S5A, inset red arrows). The Tpbpa-cre allele is inherited from the sire so the only tissues that could express cre are embryonic, fetal, or placental and in situ hybridization to detect Tpbpa mRNA in the mouse placenta at E14 shows signal exclusively in the Jz. 34 Activation of the PERK pathway maintains ER function and is part of a homeostatic response, and loss of Perk leads to hypersensitivity to ER stress. 35 The degree of placental ER stress under normal physiological conditions is minimal and there were no differences in litter size, embryonic loss, placental weight, fetal weight and placental efficiency between Perk fl/fl (control) or Jz-Perk À/À litters ( Figures S6A and S6B).
There is a close relationship between hypoxia and ER stress 36 and we previously demonstrated that hypoxia specifically activates the PERK arm of the ER-UPR pathway in both in vitro and in vivo models. 37 iScience Article Therefore, to cause ER stress we housed all pregnant females in 13% O 2 from embryonic day 0.5 (E0.5) to E18.5.
Housing under reduced oxygen resulted in increased phosphorylation of EIF2a in Jz of Perk fl/fl placentas, but this was reduced in the Jz-Perk À/À placentas while there was no difference in GRP78 and spliced variant of XBP1 protein ( Figure S7A). Using electron microscopy, we demonstrated a loss of homeostatic regulation in the ER in the Jz-Perk À/À placentas, with ER cisternae dilated only in spongiotrophoblasts ( Figure 4A, arrowheads). Protein glycosylation was also perturbed as shown by staining with Datura stramonium lectin (DSL). This binds to GlcNAc oligomers and anomalous glycoprotein aggregates/deposits accumulated exclusively in the Jz of the Jz-Perk À/À placentas ( Figure 4B, green arrows) and were clearly visible in electron micrographs of the Jz of the Jz-Perk À/À placenta ( Figure S7B, arrowheads). Furthermore, we detected aggregates showing positive staining with two other lectins (Concanavalin A, Con A, and Pisum sativum Agglutinin, PSA) which both recognize a-linked mannose, present either as part of a core oligosaccharide (Con A) or as an a-linked mannose-containing oligosaccharide (PSA, Figure S7C, arrows). This implies a potential change in glycan structures with enriched oligomannose of the glycoproteins, indicating mis-glycosylation, synthesized by the spongiotrophoblasts under ER stress. A similar pattern of lectin iScience Article staining and dilated ER cisternae was also found in the Jz of Eif2s1 tm1Rjk mutant placentas, which also display ER stress exclusively in the Jz. 30 In females housed under reduced oxygen, the litter size (ie the number of both live and dead fetuses) did not differ between females carrying either Perk fl/fl or Jz-Perk À/À litters (median(range): 7 (5-13) vs 7.5 (5-11)), Figure S7D). Females carrying Jz-Perk À/À placentas had a non-significant change in resorption rate compared to females carrying control Perk fl/fl placentas (13.3% increase, p = 0.131, Figure 4C). There was a trend to an increase in the weight of the Jz-Perk À/À placentas (6.8% higher, p = 0.086) but no change in fetal weight ( Figure 4D). The placental efficiency showed a non-significant reduction (8.0%, p = 0.228, Figure 4E). These results show that while placental ER stress in vivo causes intracellular aggregation of proteins in spongiotrophoblasts, it had little impact on placental growth, placental efficiency, and the frequency of embryonic loss.

Perturbation of placental endocrine function causes maternal maladaptation in hepatic glucose metabolism and induces cellular stress in the liver
The liver plays a crucial role in maternal metabolism, including increasing glucose and lipid availability near term to support fetal growth and in preparation for lactation. 39 We, therefore, tested whether the normal physiological adaptations occurred in the liver of pregnant females carrying litters of Jz-Perk À/À placentas compared to Perk fl/fl placentas. Maternal blood glucose concentration was measured and liver tissue harvested at E18.5. In pregnancies unaffected by ER stress (i.e. housed under atmospheric oxygen) there was no difference in maternal blood glucose concentration between the groups ( Figure S6C). In contrast, under Jz ER stress-inducing conditions, maternal blood glucose concentration was reduced by 14.1% (p = 0.024) in females carrying Jz-Perk À/À placentas compared to control Perk fl/fl placentas ( Figure 5A). Hepatic glycogen content increased by 73.3% (p = 0.035) ( Figure 5B), suggesting potential maternal hepatic maladaptation in females carrying Jz-Perk À/À placentas.
Both the AKT-GSK3 and ERK1/2 signaling pathways are central for the regulation of hepatic glycogen metabolism upon insulin or growth factor stimulation. 40 There was a 4.6-fold (p = 0.005) and 6.5-fold (p = 0.001) increase in the ratio between phosphorylated and total glycogen synthase kinase 3 a/b (GSK3a/b) and extracellular-regulated kinases (ERK1/2) respectively in females carrying Jz-Perk À/À placentas ( Figures 5C, S8, and S9A). ERKs and GSK3s respectively are activated and inactivated by phosphorylation and this promotes hepatic glycogen synthesis. 40 Phosphorylation of PDK1 and AKT, upstream kinases regulating GSK3 phosphorylation, was increased while there was no change in the phosphorylation status of 4E binding proteins (4EBP1) ( Figure S8). This suggests that whatever signals the maternal liver is receiving, they are likely promoting glycogen synthesis rather than protein translation, cell growth, and proliferation. To conclude, these results indicate that loss of correct placental signals results in maternal liver maladaptation and disruption of glucose homeostasis during pregnancy.
Anomalous hepatic glucose metabolism could impact on the maternal hepatic energy supply. Therefore, the activity of the energy-sensing kinase AMP-activated protein kinase a (AMPKa) was investigated. Indeed, AMPKa phosphorylation was markedly elevated (over 100-fold, p = 0.001) in the liver of females carrying Jz-Perk À/À placentas ( Figures 5D and S9A). As expected, the energy deprivation was associated with cellular stress in the liver as indicated by a 2-fold (p = 0.011) and 2.7-fold (p = 0.001) increase of stress kinases, p38 kinase and c-Jun N-terminal kinases (JNKs) phosphorylation respectively ( Figures 5E and S9A). Some markers of the unfolded protein response for mitochondria and ER were also increased significantly ( Figure S9B). These differences between females carrying Jz-Perk À/À and Perk fl/fl placentas could not be accounted for by litter size ( Figure S7D).
Finally, we also observed a 7.3-fold (p = 0.001) increase of DNA methyltransferase 3A (DNMT3A) in the liver of females carrying Jz-Perk À/À placentas (Figures 5F and S10A). Other epigenetic regulators (DNMT1 and TET1) were undetectable in adult liver and MECP2 was unchanged in the two groups ( Figure S10B). DNMT3A is crucial in facilitating de novo DNA methylation in somatic cells. 41  iScience Article Placental proteins are aberrantly glycosylated in early-onset pre-eclampsia Placental ER stress is a feature of early-onset pre-eclampsia 23,26 so we next sought evidence that the ER stress-mediated protein mis-glycosylation occurs in this condition. We first investigated whether placentally derived proteins in the maternal circulation of patients with pre-eclampsia are mis-glycosylated. We collected gestational age-matched maternal serum from women who remained normotensive (normal controls, NC) or went on to subsequently develop early onset pre-eclampsia (ePE), with samples collected at 28 G 1.9 wkGA (NC) and 28 G 2.4 wkGA (ePE) mean G SD, n = 5 in each group, Table S8). We hypothesized that changes in glycan structures illustrated in Figure 1A would alter their binding affinity to lectins. Therefore, to identify serum proteins with different glycosylation patterns we used wheat germ agglutinin (WGA) affinity chromatography to enrich glycoproteins before conducting isobaric Tandem Mass Tags (TMT)labeled quantitative LC-MS/MS analysis. This lectin which binds the sugar N-acetyl-D-glucosamine (GlcNAc) with preferential binding to dimers and trimers (it also has some affinity to sialic acid). We identified 113 proteins present in 9 out of 10 samples with at least 2 different peptides detected (Table S9). Seven of these were members of the pregnancy-specific glycoprotein (PSG) family. The PSGs are highly abundant glycoproteins secreted by the placenta. 42 It has been suggested that they may regulate maternal immune and vascular function and that they may have autocrine and paracrine functions. 43 There was a considerable spread of the measured values of the circulating PSGs. Of note, PSG7 was either very high or very low ( Figure 6A). This is consistent with the bimodal levels of PSG7 mRNA in the term human placenta ( Figure S11). 44 However, the copy number of the genes in the PSG locus is highly variable and one PSG7 splice variant undergoes nonsense-mediated decay. These, and other mechanisms, likely contribute to iScience Article the heterogeneity in the levels of PSG protein and mRNA. 43 Circulating PSG5 and PSG9 were lower in the ePE sera (58%, p = 0.016 and 60%, p = 0.064 respectively) compared to NC sera, while other PSGs were unchanged ( Figure 6A). PSG5 has four predicted N-linked glycosylation sites at asparagine residues N104, N111, N175, and N210 (UniportKB-Q15238). After prolonged gel electrophoresis of serum proteins, we detected multiple immunoreactive PSG5 bands, the pattern differing between NC and ePE sera ( Figure S12A). It should be noted that definitive determination of anti-PSG antibody specificity is challenging 43 However, the antibody used here was selected as being targeted against a region of PSG5 that is not glycosylated (amino acids 287-336). The other most closely related PSGs (PSG3, PSG2, and PSG11) have 81.6%, 75.5%, and 63.3% identity respectively in this region. The level of these PSGs in WGA-purified serum was unchanged when using an antibody-independent assay ( Figure 6A) suggesting that cross-reactivity, as might be seen in Figure S12C is unlikely. However, this possibility cannot be conclusively ruled out. The predicted molecular weight of nonglycosylated PSG5 is 38 kDa; however, the apparent molecular weights of the PSG5 bands ranged from $35 to $50 kDa, indicating the protein contains a range of glycans at its multiple glycosylation sites. Western blot analysis of serum PSG5 (without WGA-affinity purification) was consistent with the result obtained by mass spectrometry with serum PSG5 being lower in ePE (37.9%, p = 0.019). It is worth noting that the circulating placentally derived mis-glycosylated glycoproteins without ''protective'' sialic acid end cap and/or with high oligomannose are likely to be cleared from the circulation. 45 However, analysis of the immunoreactive bands corresponding to proteins with more, or less, complex glycans showed that the biggest change was in the loss of PSG5 which has more complex glycan side chains, (50.2%, p = 0.002, Figure 6B). In contrast, the concentration of PSG5, which has less complex side chains, remained relatively constant. To further investigate the mis-glycosylation of PSG5 as a result of placental ER stress, we collected placentas from women with ePE and normotensive term controls (Table S10). Western blot analysis showed multiple PSG5 bands indicating differences in glycosylation ( Figure S12B). The bands with higher molecular weight were predominantly present in the NTCs ( Figure S12B), consistent with the reduced complexity of PSG5 glycan we observed in ePE serum. The results using BeWo cells suggested that ER stress reduces sialylation and glycan complexity and that mis-glycosylation of PSG5 could expose more glycans with terminal N-acetylglucosamine (GlcNAc). Therefore, the same placental tissue lysates were subjected to WGA affinity chromatography to isolate PSG5-containing glycans with terminal GlcNAc prior Western blotting analysis ( Figure S12C). To overcome the unavoidable difference in gestational age of placental samples between NTC (39 G 0.8 weeks) and ePE (27 G 2.1 wk) (Table S10) we calculated the ratio between WGA-enriched and total PSG5 in placental lysates. There was an $2-fold (p = 0.007) increase in the ratio (WGA/Total) of PSG5 in the ePE placentas ( Figure 6C). These results confirm that ePE placentas that suffer from ER stress synthesize and secrete mis-glycosylated glycoproteins into the maternal circulation.
Due to the importance of sialylation for the stability of circulating glycoproteins, we investigated gene expression of all fifteen human sialyltransferases involved in the sialylation of both N-and O-linked glycosylation 31 by quantitative RT-PCR (RT-qPCR) in NTC and ePE placentas (9 per group). Five of the mRNAs were significantly reduced (ST3GAL1 Y 22%, p = 0.027, ST3GAL4 Y 52%, p = 0.042, ST3GAL6 Y 30%, p = 0.003, ST8SIA3 Y 57% (p = 0.027), ST8SIA4 Y 44%, p = 0.032) while two of the mRNAs show a trend (ST3GAL3 Y 27%, p = 0.053) and no change (ST3GAL5, p = 0.297) respectively ( Figure S13A). The remaining eight (ST3GAL2, ST6GAL1, ST6GALNAC1, ST6GALNAC2, ST6GALNAC4, ST8SIA1 and ST8SIA2 and ST8SIA5) had very low expression (>34 PCR cycles) in the placenta. Western blotting showed ST3GAL6 was reduced by 56% (p = 0.016) ( Figure S13B). However, all these data are unavoidably confounded by gestational age differences at the time of placental collection. Therefore, we investigated sialyltransferase expression in RNA-Seq datasets from BeWo cells after 24 h repetitive hypoxia-reoxygenation (HR) challenge (HR-induced injury is a potent inducer of ER stress and is central to placental pathophysiology in ePE 26,46 ). We have demonstrated that this rHR model recapitulates the changes in ER and mitochondrial UPR signaling pathways observed in ePE placentas. 26,47 The expression of three of the five sialyltransferases down-regulated in the ePE placentas was significantly reduced in rHR-treated cells -ST3GAL4 (Y35.5%, p = 0.001), ST3GAL6 (Y13.5%, p = 0.005), and ST8SIA4 (Y58.5%, p < 0.0001) while for ST3GAL1 there was a non-significant reduction (Y7.4%, p = 0.073) ( Figure 6D). ST8SIA3 was only expressed a very low level with fewer than 2 counts. This suggests that placental stress in ePE is the likely cause of downregulation of these three sialyltransferases.
Reduced expression of sialyltransferases prevents or reduces the correct addition of the sialic acid ''protective shield'' on these proteins. This loss would compromise their circulating half-life and possibly their ability to correctly signal to the maternal tissues/organs. The reduction of maternal serum PSG5 concentrations while placental WGA-binding PSG5 is elevated in ePE is consistent with this rationale. Indeed, glycoproteins with glycans carrying terminal GlcNAc are selectively cleared from the circulation in both humans and monkeys. 48 In summary, our results reveal that the glycosylation profile of placentally derived glycoproteins is altered in placentas from cases of early-onset pre-eclampsia.

DISCUSSION
During pregnancy, placental hormones modulate maternal metabolism to support fetal growth and lactation. In this study, we hypothesized that placental ER stress causes aberrant glycosylation and loss of functional activity of some of the signaling hormones and this compromises maternal adaptations to pregnancy. Our new transgenic animal model of ER stress specific to the placental endocrine zone demonstrated an association between the alteration of normal placental signals and compromised maternal hepatic glucose metabolism. The maladaptation led to increased cellular stress in the maternal liver and increased hepatic DNA methyltransferase (DNMT3A) expression, which in turn could result in epigenetic changes ( Figure 7). Furthermore, we show mis-glycosylation and reduced concentrations of secreted placental glycoproteins in pregnancies complicated by early-onset pre-eclampsia. Our results, therefore, provide the first potential mechanistic link between pregnancy complications associated with placental ER stress and adverse maternal long-term health.
N-Glycan biosynthesis is initiated in the ER and completed and matured in the Golgi apparatus through the actions of a series of glycotransferases and glycosidases. 49 For the biosynthesis, a mature N-glycan ll OPEN ACCESS iScience 26, 105911, January 20, 2023 iScience Article precursor Glc 3 Man 9 GlcNAc 2 -P-P-Dol is first synthesized in the ER before transfer to the asparagine residue in receptive Asn-X-Ser/Thr sequons in newly synthesized proteins. A trimming process on this 14-sugar glycan follows the formation of Man 8 GlcNAc 2 -Asn before the translocation of the protein to the Golgi apparatus for further glycan modification. N-glycan biosynthesis is only completed and matured by ''capping'' reactions, in which sialic acids, fucose (Fuc), galactose (Gal), and/or N-acetylglycosamine (GlcNAc) are added to the complex N-glycan branches. Capping sugars facilitate the presentation of terminal structures to lectins and antibodies. 49 Our RNA-Seq result revealed that ER stress alters the expression of 66 genes (FC > 1.5) involved in protein glycosylation (GO:00006486) and affecting almost every step of N-glycan biosynthesis ( Figure S4). GSEA further revealed the potential alteration of the trimming process of the precursor 14-sugar glycan by ER stress. As the precursor glycan is central for subsequent glycan oligomannose and complex glycan formation, this finding suggests that ER stress not only induces misglycosylation, but may also reduce overall protein glycosylation. This is supported by increased RNA from genes involved in protein deglycosylation ( Figure S3B). Indeed, we observed a reduced degree of glycosylation of hCGb and VEGFA under Tg treatment. ER stress-mediated alterations of glycan structures have been reported using genetic manipulation or the ER stress inducer thapsigargin (Tg), 22 which also revealed that the induced glycosylation change affects both secreted and membrane protein glycosylation. 22 This finding is consistent with changes in lectin staining in placentas from cases of pre-eclampsia, 50,51 where ER stress-mediated alteration of placenta protein glycosylation may affect the activity of placentally derived proteins and the glycans arrayed on membrane receptors, carriers, and transporters on the syncytiotrophoblast and endothelial cells. These could have profound effects on ''self-nonself'' discrimination during pregnancy, receipt of correct maternal signals, and nutrient exchange. Interruption of this twoway communication between the mother and placenta could also have significant effects on maternal physiological adaptations and placental function, resulting in poor pregnancy outcomes.
The composition of the glycocalyx covering the placenta is changed in cases of pre-eclampsia, 50,51 with an increase in oligomannose and a decrease in sialic acids in both syncytiotrophoblast and fetal endothelial cells. 51 Reduced structures with a-2,3-linked sialic acid in placentas from ePE is consistent with the iScience Article downregulation of sialyltransferases ST3GAL4 and ST3GAL6 and exposes glycans with terminal galactose. On the other hand, the increase of WGA-isolated placental PSG5 also indicates that the ePE placenta synthesize glycoproteins containing high level of glycans with terminal GlcNAc, rather than sialic acid. Sialylation protects glycoproteins from glycosidases and proteases, 18 thus increasing their circulating half-life. In the liver, asialoglycoprotein receptors (Asgrs) on hepatocytes and the mannose receptor (ManR) on sinusoidal endothelial cells and Kupffer cells are two conserved receptors involved in the clearance of circulating glycoproteins with exposed terminal galactose structures due to lack of sialic acid end-caps and containing high mannose N-glycan respectively. 52 These receptors also play a central role in the clearance of placentally derived factors with aberrant glycosylation. 45 Additionally, glycoproteins with terminal GlcNAc can also be selectively cleared from the circulation by the clearance receptor(s), which has not yet to be identified. 48 However, an in vitro study shows the glycoproteins with terminal GlcNAc can bind to ManR 53 and this increased clearance could explain why the concentration of PSG5 which contains high terminal GlcNAc, was reduced in ePE serum. Crucially, our results demonstrated that the glycosylation change in the ePE placenta is consistent with ER stress. Using the Jz-Perk À/À model, we further showed that correct placental signals are necessary for normal maternal hepatic glucose metabolism and avoidance of cellular stresses induced by the physiological changes during pregnancy.
Mounting epidemiological evidence links pregnancy complications to an increased maternal risk of metabolic disease in later life. [5][6][7] Lack of the correct placental signals that normally induce changes in maternal organs to meet fetal demands could overwhelm homeostatic mechanisms and induce cellular stress, thereby causing irreversible damage. The increase in hepatic Dnmt3a mRNA, encoding a DNA methyltransferase, may implicate de novo DNA methylation in the liver cells under stress. 41 A change in liver DNA methylation of genes relevant to the development of diabetes is seen in patients with type II diabetes. 54 A post-partum mouse study will be necessary to test links between the genome-wide DNA methylation pattern in livers from females carrying Jz-Perk À/À placentas and signs of diabetes in later life.
The placenta also releases other factors (cytokines, chemokines, and so forth) not analyzed in the course of this work. It is likely that the release of these may also be altered by placental ER stress. These could also affect maternal adaptation so would be an area for future study.
To conclude, we show how placental stress can lead to maternal physiological maladaptation to pregnancy by causing aberrant glycosylation of placentally derived factors. Alleviating this stress, therefore, has the potential to improve long-term maternal health. We recently showed that tauroursodeoxycholic acid (TUDCA), a biliary salt, greatly reduces chronic placental ER stress. 55 As the related ursodeoxycholic acid (UDCA) has already been used in the treatment of intrahepatic cholestasis during pregnancy, 56 interventions based on this chaperone are worth further study.

Limitations of the study
This study has four main limitations; i) We generated several lines of evidence, albeit indirect, that show placental cells when exposed to ER stress secrete aberrantly glycosylated proteins and that misglycosylated protein looses its normal biological activity. Nevertheless, our results are consistent with the study by Wong et al. 22 but direct evidence would be stronger. ii) In the placental-specific ER stress (Jz-Perk À/À ) animal model, we had to use mild hypoxia to exacerbate ER stress in the placenta. Although Perk fl/fl animals were also exposed to hypoxia, we cannot exclude possible interference of hypoxia on maternal physiology. To overcome this we are developing another animal model with placental ER stress without the requirement for an additional stressor. iii) The placental-specific ER stress (Jz-Perk À/À ) animal model has defective maternal mal-adaptation of hepatic glucose metabolism. However, the detail of which placental protein(s) mediate this and mechanism underlying this are not known. iv) While we demonstrated the misglycosylation of placental proteins in human pregnancy, it is not known whether the metabolic maladaptation we observed in mice also occurs in humans.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank Emma Eastwell and all animal technicians in Combined Animal Facilities for animal care. We also thank the staff of the Cambridge Advanced Imaging Center for electron microscopy and the Cambridge Proteomic Center for proteomic analysis. Finally, we thank the team at Upsala University and the research midwives and assistants from the Department of Women and Children's Health at Guy's and St Thomas' Foundation Trust for collecting blood and placental samples and associated clinical data. This work was supported by BBSRC grant BB/K016164/1 (to S.M.H. and A.D.) and through the National Institute for Health Research Comprehensive Biomedical Research Centre Award to Guy's & St. Thomas' NHS Foundation Trust, in partnership with King's College London and King's College Hospital NHS Foundation Trust (to C.G., K.D., and L.C.). All other work was supported by the Wellcome Trust Grant 084804/2/08/Z and MRC Confidence in Concepts award 2019 R8.6. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health and Social Care. The Graphical Abstract was generated using Biorender.com.

AUTHOR CONTRIBUTIONS
HWY conceived, designed, directed, and performed experiments, collected, analyzed, and interpreted experimental data; prepared figures and drafted the article. XZ performed RNA-Seq data analysis and prepared corresponding graphs and data for deposition in public repositories.
LG and CB performed Western blots. PCP performed the glycomic analysis of conditioned media. CJPJ conducted lectin immunostaining. CG, KD, and MO collected serum and placental clinical materials. LC managed the collection of serum samples and critically reviewed the article. SMH and AD analyzed glycomic data, interpreted experimental data, contributed to writing, and critically reviewed the article. GJB and DSC-J analyzed and interpreted experimental data, contributed to writing and critically reviewed the article. All authors revised and gave final approval to the article.

DECLARATION OF INTERESTS
Authors declare no competing interests.

Genotyping of transgenic animals
For genotyping, an ear biopsy or a small piece of embryonic tissue was used. The tissue was lysed in Direct PCR Lysis Reagent (102-T for tissues or 402-E for ear biopsies, Bioquote Ltd, UK) containing 1 mg/mL proteinase K (P6556, Sigma UK) at 55 C for 2 h followed by 85 C for 45 min. 1 mL was used for PCR and PCR products were resolved at 2% agarose gel. For primer sequences, see Table S11.

Cell and tissue culture
All culture reagents were purchased from ThermoFisher Scientific, UK unless otherwise stated.

Jz explant culture
The placenta from E12.5 was dissected into decidua, junctional and labyrinth layers. The junctional layer was cut into 1 mm 3 pieces and placed on a 40 mm cell strainer submerged in a 6 well plate. Jz explants were cultured for 48 h in a humidified 37 C incubator at 10% O 2 in defined serum-free medium comprising: advanced DMEM/F12 reduced serum medium (12634), 1X GlutaMax (35050061), 1X penicillin/streptomycin (15140-122), 0.5X B27 supplement (17504044). Thapsigargin (100 nM, Sigma, UK) was added to some cultures. Conditioned medium was harvested and centrifuged at 4000 rpm (1771 g) for 30 min at 4 C to remove tissue debris before concentration using Vivaspin (5000 MWCO) centrifugal concentrator (Vivaproducts Inc, USA) according to manufacturer's instructions. Protein concentration was determined using the BCA (Sigma, UK).

Mouse embryonic fibroblast isolation
Mouse embryos were collected at E12.5. The remainder of the embryo was incubated with 0.05% Trypsin/ EDTA (11580626) overnight at 4 C. The Trypsin was replaced with 1 mL of fresh 0.05% Trypsin/EDTA and incubated for 15 min at 37 C with occasionally swirling. An equal amount of DMEM (41966) containing 10% FBS (10270106), 1X penicillin/streptomycin was added and tissue dissociated by gentle pipetting. The suspension was sieved through a 40 mm mesh to remove all aggregates before centrifugation. The pellet was resuspended in 10% FBS containing DMEM medium and the cells were cultured in a humidified 37 C incubator with 5% CO 2 .

OPEN ACCESS
iScience 26, 105911, January 20, 2023 23 iScience Article Human choriocarcinoma BeWo cells (ATCC, CCL-98) were cultured in modified DMEM/F12 medium containing 5.5 mM glucose, 10% FBS and 1X penicillin/streptomycin in a humidified 37 C incubator with 5% CO 2 . For all experiments and glycomic analysis cells were rinsed with serum free medium and then cultured for 24 h with Tg (100 nM) or vehicle (0.1% DMSO) in serum free medium. Conditioned medium was harvested, debris removed by centrifugation and stored at À80 C until use. Cells were not syncytialised as this can be somewhat variable and this would introduce experimental noise.

VEGFA stimulated VEGFR2 phosphorylation
Human umbilical vein endothelial cells (HUVECs) were kindly provided by Dr Cindrova-Davies (University of Cambridge). 68 To assay VEGFA activity, HUVECs were pre-treated with serum-free medium for 6 h. The cells were then incubated with conditioned media, medium+25 ng/mL recombinant VEGFA (Sigma-Aldrich, UK), or medium alone for 10 min. HUVEC were harvested as described below and lysates stored at À80 C until Western blot analysis of the phosphorylation status of VEGFR2.

Western blotting
Western blotting was performed as previously described. 47 Details of all antibodies are provided in Table S12.
Glycoprotein enrichment by lectin affinity chromatography, WGA Glycoproteins were enriched from placental lysates or albumin-depleted sera using the Pierce Glycoprotein Isolation Kit, WGA (89805, ThermoFisher Scientific, UK) according to manufacturer's instruction. The glycoproteins were eluted directly either with 1X gel loading buffer and boiled at 70 C for 10 min before Western blot analysis or with elution buffer for proteomic analysis.

Proteomic analysis
Two-dimension fluorescence Difference Gel Electrophoresis (2-D DIGE) of Jz explant conditioned media DIGE and MS analyses of conditioned media harvested from the Jz explants were carried out in the Cambridge Center for Proteomics. 69 Equal amounts of protein were mixed with Chaps/Thiourea buffer containing 6 M Urea, 2 M Thiourea, 4% Chaps, 10 mM Tris (pH 8), vortexed and sonicated to solubilise. pH was adjusted to pH 8.5 with 50 mM NaOH. Samples were quantified using Quick Startä Bradford (Bio-Rad). Samples were then labeled with Cy5 dye and pooled with Cy3 dye according to the manufacturer's instructions using Chromis DGE Minimal Labeling Kit (Cyanagen, Bologna, Italy) and incubated for 30 min. The reaction was quenched by adding 10 mM lysine and incubated for 10 min.
For IEF, the sample was soaked on 3-10NL Immobiline Dry Strip Gel (GE Healthcare). First dimension IEF focusing was performed with the Protean i12 IEF Cell (Bio-Rad) using the following parameters: 20V for 10 h, 500V for 1 h, 1000V for 1 h, 8000V until 40000 Vhrs was achieved. After IEF, the strips were equilibrated in a buffer containing 100 mM Tris (pH 6.8), 30% glycerol, 6 M Urea, 2% SDS and 1% DTT (freshly added) for 15 min following by 15 min in a buffer using the same composition except that 1% DDT was replaced with iodoacetamide 2.5%. The second dimension was then resolved in 12% SDS-polyacrylamide gel.
Gels were scanned using a Typhoon 9400 laser scanner (GE Healthcare) and analyzed using mageMas-ter2D, DeCyder 5.02 version software (GE Healthcare). Differential spots were identified, one of the gels silver stained and the spots of interest matched from the DeCyder image of the same gel to the silver stained image. Spots were cut manually and destained, reduced, alkylated and digested prior to LC-MS/MS analysis (see below).
TMT isobaric mass tags labelling of serum proteins Serum albumin was depleted from both NC and ePE sera using ProteoPrepâ Blue Albumin & IgG Depletion Kit (PROTBA-1KT, Sigma) following manufacturer's instructions except tris-buffered saline (TBS) was used as equilibrium buffer. Protein concentration in albumin-depleted serum sample was measured using the BCA.